Chapter 8 – Bacterial and Viral Genetic Systems

Bacteria• Prototrophic

– Wild-type– Can grow on minimal media

• Contains minimal nutrients – carbon, nitrogen, phosphorous, vitamins, ions

• Auxotrophic– Can not produce an essential enzyme or manufacture

essential molecules– Will only grow on media that contains the “missing”

substance • Complete media

Culturing bacteria• Suspension culture

– Liquid media

– Bacteria dies off when nutrients are used up or waste buildup becomes toxic

– Bacteria grow singularly – no colonies

Culturing bacteria

• Petri dishes

– Growth media in agar

– Isolate individual colonies

• Each colony originates from a single bacterium

Replica plating• Gives “carbon

copies” of petri dish colonies

• Use sterilized velvet to make a stamp– Some bacteria

from each colony is transferred to velvet, and then transferred to new dishes

Bacterial genome

• Most consist of a single, circular chromosome– Some have several

chromosomes, and a few have linear chromosomes

• Very little “extra” DNA between genes

• Plasmids – Small, circular, extra-

chromosomal DNA• Usually non-essential

– Replicate independent of chromosomal DNA

• Have their own origin of replication

F factor episome

• Episome– A plasmid that

can replicate independently AND also has the ability to incorporate into chromosomes

Gene transfer in bacteria - Conjugation• One bacteria directly transfers DNA to another bacterium

• Cytoplasmic connection forms, and either entire plasmid or part of the chromosome is transferred from donor to recipient

• Crossing over may occur between homolgous regions– Creates recombinant DNA– Extra DNA is degraded

Gene transfer in bacteria - Transformation

• Bacteria takes up DNA from surrounding environment

• Recombination may occur

Gene transfer in bacteria - Transduction

• Viral particle introduced DNA from a bacterium into a new bacterium

Conjugation• Fertility factor/F factor contains ori and genes needed for

conjugation

Conjugation• Hfr bacteria

– F+ cell that has F factor incorporated into chromosome

• As F factor enters recipient, some chromsome enters – amount depends on time length of contact

• Donor DNA made into double-stranded– Crossing over can occur

between homologous regions

– Any DNA not incorporated is degraded

• Recipient is not usually converted to F+ since the F factor is nicked in the middle

Conjugation• F′ bacteria

– F factor excises out of a chromosome in a Hfr cell• May remove part of chromosome as well

– F′ plasmid now contains F factor and some genes from chromosome• Enters F- bacteria

– Produces merozygotes – partially diploid

Transformation• Uptake of DNA and incorporation into chromosome or plasmid

• Competent – cells able to take up DNA– CaCl2, heat shock, electrical fields

• Makes membrane more permeable to DNA– DNA does not have to have bacterial origin

• Transformants – bacteria that have incorporated foreign DNA

E. Coli has model organism• Many strains are avirulent

• Small and rapid reproduction

• Easy to culture

• Genome is single chromosome - haploid– Small genome– 4.5 million bp/4,000 genes

• Wild-type are prototrophic

Viral genetics• DNA or RNA

(single or double stranded) as genetic material

• Can not reproduce on their own

• Bacteriophages – viral particles that infect bacteria

Bacteriophage – lytic cycle• Virulent phages

• Viral DNA is injected into host cell where it replicated, transcribed, and translated into more phages

• Host cell bursts open to release viral particles

Bacteriophages – lysogenic cycle• Temperate phages

• Phage DNA is incorporated into host genome – prophage

• Passed onto all progeny cells

• Can be transcribed and translated

• Can exit from host genome to enter lytic cycle

Transduction• Generalized

– Any gene is transferred– During lytic cycle, bacterial DNA is degraded

• Some may enter viral protein coat instead of viral genetic material – Transducing phages

• Can become incorporated into new host’s genome

Transduction• Specialized

– Few genes are transferred/genes near certain sites of chromosome

– During lysogenic cycle, prophage enters at specific sites of host’s genome

– When prophage excises, it may do so imperfectly and bring some hot DNA with it

• Then introduced to new host

RNA viruses• Positive strand RNA viruses

– Single strand directly codes for viral proteins

• Negative strand RNA viruses– Must make complementary RNA strand, which then codes for proteins

• Retroviruses– Incorporate into host genome

• Must make DNA from RNA• Reverse transcriptase

– Makes cDNA from DNA or RNA template

– Enters host genome as a provirus• Can be transcribed and translated

– Some retroviruses contain oncogenes• Cause tumors